Earlier Antenatal Diagnosis of Hemoglobinopathies By Coelocentesis

In countries with a high prevalence of hemoglobinopathy carriers, the only realistic approach to control the birth of new patients with thalassemia major or sickle cell disease is population screening in combination with invasive prenatal diagnosis [1].   In the early 1990's, molecular definition of the thalassemia defects, development of procedures for their detection by DNA analysis and introduction of amniotic fluid sampling (amniocentesis) or chorionic villus sampling (CVS) led to early prenatal diagnosis at 16 and 11 weeks, respectively [2].  An alternative technique for earlier diagnosis is celocentesis [3].  At the end of week 4 of gestation, the developing exocoelomic cavity (ECC) splits the extra-embryonic mesoderm into two layers, the somatic mesoderm lining the trophoblast and the splanchnic mesoderm covering the secondary yolk sac and the embryo. The ECC is the largest anatomical space inside the gestational sac between 5 and 9 weeks of gestation and is  surrounded by celomic fluid (CF), which contains cells of fetal origin [4-5]. This fluid can be sampled by a technique that involves the ultrasound-guided insertion of a needle through the vagina from as early as 7 weeks  of gestation. Previous studies utilizing coelocentesis for prenatal determination of  single-gene defects reported variable success ranging from 58 to 95% because of presence of maternal cell contamination (MCC) [6].  In this work we demostred as this problem  can be   overcome through the identification of embryo-fetal erythroid precursors  presented in the coelomatic fluid and their  specific selection. 302 couples from different regions of Italy, at risk for ß thalassemia or sickle cell disease  asked for prenatal diagnosis by coelocentesis  that was carried out at between 6+6 and 9+2 weeks' gestation. Coelomic fluid samples with no or very low (<5%) maternal contamination (100 samples)  were successfully analyzed without preliminary treatment. In samples with >5% maternal contamination, two different procedures were used to isolate embryo-fetal cells:  the first technique involved positive selection of embryo-fetal erythroid precursors by anti-CD71 MicroBeads (160 samples), the second procedure  was through the use of a micromanipulator (42 samples). In 68/300 (22.6%) cases the fetus was affected by ß-thalassemia major or sicke cell / ß-thalassemia and 66 women chosen to terminate the pregnancy. Two families decided to continue the pregnancy for ethical reasons despite the documented presence of an affected fetus. The antenatal diagnosis was confirmed in all 66 cases by molecular analysis of placental tissue after termination (Table 1). In 232/300 (77.4%) cases the fetus was diagnosed as being normal or a carrier for ß-thalassemia or sicke cell disease. The results obtained after coelocentesis were confirmed by amniocentesis or postnatally. In two case (0.66%) was no obtained a reliable diagnosis because no fetal cells were found (Table 1).         The findings of this study in a large number of pregnancies investigated by coelocentesis, demonstrate that embryo-fetal erytroid cell selection from coelomatic fluid allows reliable and earlier prenatal diagnosis of hemoglobinopathies.  This technique is attractive to parents because it provides  prenatal diagnosis of genetic disease at least 4 weeks earlier respect to traditional procedures reducing anxiety of parents and from a clinical point of view this procedure  would allow women to undergo medical TOP at 8–10 weeks of gestation which is less traumatic and safer than second trimester surgical TOP. 

 

Table 1. Overall results of prenatal diagnosis of  hemoglobinopathies by coelocentesis.

 

Number of coelocentesis effectuated	302
Results of Prenatal diagnosis:
Affected	68
Carrier of hemoglobinopathies	161
Non Affected	71
diagnosis not reiable	2
Control post coelocentesis:
Placental tissue after termination	66
CVS or amniocentesis	153
Postnatal test	61
Awaiting	20
Coelocentesis diagnostic errors	0